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Transcription activator-like effector nuclease-mediated transduction of exogenous gene into IL2RG locus.

ABSTRACT: X-linked severe combined immunodeficiency (SCID-X1) caused by mutations in interleukin 2 receptor gamma (IL2RG) gene threatens the survival of affected boys during the first year of life unless hematopoietic stem cell transplantation is provided. Although viral vector-mediated gene therapy has been successfully performed in patients with no HLA-matched donors, leukemia caused by vector-mediated insertional mutagenesis has been reported in some individuals. Transcription activator-like effector nuclease (TALEN) is an artificial sequence-specific endonuclease that is expected to revolutionize the precise correction of disease-causing mutations and eliminate the risk of insertional mutagenesis. Here, we report TALEN-mediated genome editing of the IL2RG locus. We transfected TALENs along with a targeting vector into Jurkat cells, and we confirmed the precise introduction of the exogenous gene into the IL2RG locus. In addition, we found that the length of homology arm in the targeting vector influenced the efficiency of TALEN-mediated homologous recombination.

SUBMITTER: Matsubara Y

PROVIDER: S-EPMC4031469 | biostudies-literature | 2014 May

REPOSITORIES: biostudies-literature

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Transcription activator-like effector nuclease-mediated transduction of exogenous gene into IL2RG locus.

Matsubara Yohei Y Chiba Tomoki T Kashimada Kenichi K Morio Tomohiro T Takada Shuji S Mizutani Shuki S Asahara Hiroshi H

Scientific reports 20140523

X-linked severe combined immunodeficiency (SCID-X1) caused by mutations in interleukin 2 receptor gamma (IL2RG) gene threatens the survival of affected boys during the first year of life unless hematopoietic stem cell transplantation is provided. Although viral vector-mediated gene therapy has been successfully performed in patients with no HLA-matched donors, leukemia caused by vector-mediated insertional mutagenesis has been reported in some individuals. Transcription activator-like effector n ...[more]

PMID: 24853770

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Project description:Genetically engineered mice are a valuable resource for studies of the behavioral effects of ethanol. However, for some behavioral tests of ethanol action, the rat is a superior model organism. Production of genetically engineered rats has been severely hampered due to technical limitations. Here we utilized a promising new technique for efficient site-specific gene modification to create a novel gene knockout rat line. This approach is based on transcriptional activator-like effector nucleases (TALENs). TALENs function in pairs and bind DNA in a sequence-specific manner. Upon binding to the target sequence, a functional nuclease is reconstituted that creates double-stranded breaks in the DNA that are efficiently repaired by non-homologous end joining. This error-prone process often results in deletions of varying lengths at the targeted locus. The toll-like receptor 4 (Tlr4) gene was selected for TALEN-mediated gene inactivation. Tlr4 has been implicated in ethanol-induced neuroinflammation and neurodegeneration, as well as multiple ethanol-induced behavioral effects. To generate Tlr4 knockout rats, a pair of TALEN constructs was created that specifically target Exon 1 immediately downstream of the start of translation. TALEN mRNAs were microinjected into the cytoplasm of one-cell Wistar rat embryos. Of 13 live-born pups that resulted, one harbored a mutation in Exon 1 of Tlr4. The mutated allele consisted of a 13 base-pair deletion that was predicted to create a frameshift mutation after amino acid 25. This founder rat successfully transmitted the mutation to F1 offspring. Heterozygous F1 offspring were interbred to produce homozygous F2 animals. Homozygous mutants expressed the 13-bp deletion in Tlr4 mRNA. In contrast to control rats that produced a robust increase in plasma tumor necrosis factor alpha in response to a lipopolysaccharide challenge, homozygous rats had a markedly attenuated response. Thus, the mutant Tlr4 allele generated by TALEN-mediated gene inactivation represents a null allele. This knockout rat line will be valuable for studies of ethanol action as well as more general inflammatory conditions including septic shock. In conclusion, TALEN-mediated gene targeting in rat zygotes represents an inexpensive, efficient, and rapid method for creating genetically engineered rats.

Dataset Information

Transcription activator-like effector nuclease-mediated transduction of exogenous gene into IL2RG locus.

Publications

Transcription activator-like effector nuclease-mediated transduction of exogenous gene into IL2RG locus.

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